The etiological agent of the dengue fever is the Dengue virus, belonging to the Flavivirus genus of the family Flaviviridae (Burke and Monath, 2001). A particularly important subgroup of Flaviviruses is the group of so called mosquito-borne Flaviviruses, i.e. Flaviviruses that are transmitted by mosquitos. This group comprises in addition to the above mentioned Denguevirus other important viruses such as the West nile virus, the Japanese encephalitis virus and the Yellow fever virus (Fields Virology, ed. by Fields B. N., Lippincott-Raven Publishers, 3rd edition 1996, ISBN: 0-7817-0253-4, pages 931-1034). Typical diseases transmitted by these viruses are West nile fever and West nile encephalitis induced by the West nile virus, encephalitis induced by the Japanese encephalitis virus, Yellow fever induced by the Yellow fever virus and Dengue fever, dengue hemorrhagic fever (DHF; see below) and Dengue shock syndrome (DSS) induced by the Dengue virus.
Flaviviruses are enveloped, single-stranded, positive-sense RNA viruses formed by three structural proteins: the capsid protein (C) that forms a nucleocapsid in association with the viral genome, which is surrounded by a lipid bilayer in which are anchored the M (membrane) and E (envelope) proteins. The genome is approximately 11 kb long and contains a single open reading frame encoding a polyprotein precursor of about 3400 amino acid residues. Individual viral proteins are generated from this precursor by the action of cellular and viral proteases. The three structural proteins (C, M and E) are derived from the N-terminal part of the polyprotein and are followed by seven non-structural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 (Lindenbach and Rice, 2001).
Glycoprotein NS1, present in all Flaviviruses, appears to be essential for virus viability. Dengue virus NS1 is secreted from mammalian infected cells in a soluble hexameric form (Flamand et al., 1999). This noncovalently bound hexameric complex is formed by 3 dimeric subunits and has a molecular mass of 310 kDa. Dimerization is a prerequisite for NS1 protein export to the plasma membrane, where it remains as the unique viral resident protein of the infected cell surface.
In mammalian cells, but not in insect cell lines that support dengue infection, part of the transported NS1 is released into the extracellular milieu. Extracellular NS1 is secreted either as a soluble protein, which exist in a higher hexameric oligomeric form, or in association with microparticles but not with virions. In addition, NS1 has been found circulating in sera from dengue virus infected patients, suggesting that secretion of NS1 may be an important event in Flavivirus infection in the human host. During the course of a Flavivirus infection, the NS1 protein evokes a strong antibody response, which helps to clear the infecting virus from the host, presumably through a complement-mediated pathway (Schlesinger, J. J. et al., 1987) and antibody-dependant cell cytotoxicity (ADCC) (Schlesinger, J. J. et al., 1993).
The Dengue virus, with its four serotypes Dengue virus serotype 1 (Den-1) to Dengue virus serotype 4 (Den-4), is the most important member of the Flavivirus genus with respect to infections of humans and produces diseases that range from flu-like symptoms to severe or fatal illness, dengue haemorrhagic fever with shock syndrome. Dengue outbreaks continue to be a major public health problem in densely populated areas of the tropical and subtropical regions, where mosquito vectors are abundant.
The concern over the spread of dengue infection and other diseases induced by mosquito-borne Flaviviruses in many parts of the world has resulted in more efforts being made towards the development of dengue vaccines, which could prevent both dengue fever (DF), and dengue hemorrhagic fever (DHF) and in vaccines useful to protect the vaccinated individual against infections induced by some or all mosquito-borne Flaviviruses.
While most cases of DF are manifested after the first infection by any of the four serotypes, a large percentage of DHF cases occur in subjects who are infected for the second time by a serotype which is different from the first infecting serotype of dengue virus. These observations give rise to the hypothesis that sequential infection of an individual with antibody against one dengue serotype by a different virus serotype at an appropriate interval may result in DHF in a certain number of cases. Antibody-dependant enhancement (ADE) has been demonstrated in vitro for dengue viruses, as well as other enveloped viruses, and is considered to be an important mechanism in the pathogenesis of DHF.
It has also been observed that DHF usually emerges in geographic areas where multiple (three or four) virus serotypes co-circulate. In regions with endemic DHF such as Southeast Asian countries, the age-specific attack rate is higher in children, and the number of DHF cases decreases in higher age groups. This roughly corresponds with the increasing seroprevalence to dengue, indicating that natural infection may evoke protective immunity. This phenomenon is not unlike that observed with other viral infections such as hepatitis A virus. Anecdotal clinical observations have shown that patients may experience DHF twice (Nimmannitya et al., 1990) but this is rare, and it is difficult to identify accurately the serotypes causing the second and subsequent infections. So far, there has been no reports of a forth infection in the same individual, despite the fact that all four dengue virus serotypes circulate in the same area. This suggests that, in nature, infection by two or three dengue virus serotypes in the same individual may result in cross-reactive antibodies or even a cross-reactive cytotoxic lymphocyte response. This may modulate or protect against infection by the remaining dengue virus serotypes in nature.
At present there are no approved dengue vaccines. Today, prevention of dengue virus infection is dependent upon control of the principal mosquito vector, Aedes aegypti. Insecticide resistance, lack of technical and financial support that would enable local health departments to maintain effective mosquito control programs, and continuing geographic spread of both the vector mosquitoes and dengue viruses make it practically impossible to prevent dengue infections by current mosquito control programs. Therefore, development of safe and effective vaccines against all four serotypes of dengue virus has been designated by the WHO as a priority for the most cost-effective means to prevent dengue virus infection. The WHO has recommended that the ideal vaccine against dengue and DHF should be the kind that can prevent infection caused by all serotypes so that sequential infection cannot happen.
To this end WO 98/13500 proposes to use a recombinant Modified Vaccinia Virus Ankara (MVA) expressing antigens from all Dengue virus serotypes or to use four recombinant MVA wherein each of the recombinant MVA expresses at least one antigen of one Dengue virus serotype. Both strategies provide very promising strategies to vaccinate against all Dengue virus serotypes. However, it is desirable to provide a single subunit vaccine that upon administration results in an immune response against more than one Flavivirus or against more than one serotype of Dengue virus, preferably against all Dengue virus serotypes. Moreover, WO 98/13500 discloses a recombinant MVA encoding Dengue virus NS1. WO 98/13500 does not disclose that an antigen derived from one Dengue virus serotype elicits an immune response not only against the Dengue virus serotype from which the antigen is derived but also against antigens derived from other Dengue virus serotypes.
WO 99/15692 discloses a recombinant MVA containing and capable of expressing one or more DNA sequences encoding Dengue virus antigens not able to effect immune enhancement or antibody dependant enhancement. WO 99/15692 does not disclose that an antigen derived from one Dengue virus serotype elicits an immune response not only against the Dengue virus serotype from which the antigen is derived but also against antigens derived from other Dengue virus serotypes.